Nucleic acid sequence and protein in addition to polypeptides coding for mannitol dehydrogenases or parts thereof and the production and use thereof in diagnosis and therapy

ABSTRACT

The invention relates to polypeptides made of mannitol-dehydrogenase of  Cladosporium herbarum  and  Alternaria alternata  nucleic acids coding therefor and the use thereof in diagnosis and therapy.

In certain people, allergic reactions are caused by a wide range of substances. Not only allergies to components of animals, such as, for example, cat hairs, but also allergies to plants and plant parts, such as the pollen of flowers, are known. However, allergies to microorganisms such as, for example, molds, are also known.

The present invention relates to the major allergen of the mold Cladosporium herbarum. It has been found within the scope of the present invention that, surprisingly, this major allergen is a mannitol dehydrogenase (MtDH). The present invention furthermore relates to a major allergen of Alternaria alternata.

One aspect of the present invention relates to a polypeptide which has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 1. The amino acid sequence is shown in FIG. 1, as is the corresponding DNA sequence. The invention also relates to the polypeptides with the seq. ID No. 4, 5 and 6.

The complete sequence of a polypeptide encoding a mannitol dehydrogenase is furthermore disclosed. Seq. ID No. 7 represents the nucleic acid sequence and seq. ID No. 8 the amino acid sequence thereof. The sequences are shown in FIG. 3.

A further aspect of the present invention relates to a polypeptide which has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 11. The amino acid sequence is shown in FIG. 4; it constitutes a major allergen which has been isolated from Alternaria alternata.

The amino acid sequence of the major allergen from. Alternaria alternata, which is mannitol dehydrogenase, is shown in FIG. 5 in the one-letter code, together with the DNA sequence encoding it (sequence ID No. 12) and the flanking nucleotide sequences at the 5′ and the 3′ end.

A polypeptide according to the invention preferably has at least one epitope. An epitope is understood as meaning a region to which antibodies can bind. In principle, there are linear epitopes. In this case, the amino acids which form the epitope are arranged next to one another. However, what are known as the confirmation epitopes are more frequent. These confirmation epitopes are formed by the folding of the polypeptide. Here, amino acids which are not adjacent in the sequence can come into spatial vicinity owing to the three-dimensional folding of the polypeptide, and this surface structure is bound by an antibody.

Preferably, the epitopes are specific for one mold. In principle, antibodies against virtually all amino acid sequences can be generated with the aid of suitable techniques, for example using adjuvants. The polypeptides according to the invention, however, play an important role in diagnostics and therapy. This is why the polypeptides according to the invention preferably have specific epitopes, the epitopes being specific for one mold. Frequently, proteins or polypeptides which originate from a certain organism have similarities to a corresponding protein or polypeptide which originates from a related organism. It is therefore possible that antibodies which are directed against an epitope of a certain mold also react with a corresponding epitope of a related mold.

The more specific an epitope, the less antibodies which are directed against it will react with an epitope of a homologous polypeptide from a related organism. Thus, the polypeptides according to the invention preferably have those epitopes which are specific for a mold. The polypeptides especially preferably have those epitopes which are specific for a mold of the genus Cladosporium. The polypeptides very especially preferably have an epitope which is specific for Cladosporium herbarum. Antibodies which are directed against such an epitope do not react with other polypeptides.

It has been found within the scope of the present invention that the polypeptide with the amino acid sequence ID No. 1 encodes a mannitol dehydrogenase. The present invention also relates to parts of this amino acid sequence with at least 11 amino acids. The polypeptides according to the invention preferably have at least 20 consecutive amino acids from the sequence with the sequence ID No. 1. More preferred are those polypeptides which have at least 50 consecutive amino acids, and very especially preferred are those polypeptides which have at least 100 consecutive amino acids from the amino acid sequence with the sequence ID No. 1.

The invention also relates to a polypeptide from the N-terminus with the sequence PGQQATKHESLLDQLS (seq. ID No. 4) and to two polypeptides from the C-terminal end with the sequence LDTGLSDFVVK (seq. ID No. 5) and MGRDGLAKEL (seq. ID No. 6).

The invention also relates to a polypeptide with the sequence ID No. 8 and to parts of this polypeptide which comprise an epitope. The parts according to the invention of the sequence ID No. 8 have at least 11, preferably at least 20, more preferably at least 50 and very especially preferably at least 100 consecutive amino acids from the amino acid sequence with the sequence ID No. 8.

The present invention furthermore relates to a polypeptide with the sequence ID No. 11 and to parts of this polypeptide which comprise an epitope. Such epitopes are specific for Alternaria, more precisely for Alternaria alternata. The parts according to the invention of the sequence ID No. 11 have at least 11, preferably at least 20, more preferably at least 50 and especially preferably at least 100 consecutive amino acids from the amino acid sequence with the sequence ID No. 11.

These polypeptides preferably have at least one epitope. For example, it is possible, with the aid of hydrophilicity/hydrophobicity examinations, to identify those parts of the polypeptide which are especially suitable for immunological reactions. This can be done for example with the aid of suitable computer programs.

As an alternative, it is also possible to prepare fragments of the sequence with the aid of what is known as the Pepscan method and to test the short fragments for relevant epitopes by reacting them with sera from allergic patients. Moreover, it must be identified whether the epitopes are epitopes which are specific for a mold, in particular for a mold from the genus Cladosporium and/or Alternaria and in particular for Cladosporium herbarum and/or Alternaria alternata. This determination is carried out using suitable serum panels.

Cladosporium is a fungal genus which belongs to the molds. Cladosporium species are very frequent and occur preferentially in bogs, in forests and in gardens since they grow readily on rotten plants or on leaves. Moreover, they are found in greenhouses and in insufficiently cleaned refrigerators. Cladosporium also grows on textiles, for example linen fabrics. Cladosporium can trigger allergic reactions such as, for example, running nose, cough, sneezing, urticaria or asthma (mold allergy).

Alternaria is a fungal genus which belongs to the molds. Alternaria species occur preferentially in bogs, in forests and in gardens since they grow readily on rotten plants or on leaves. On domestic premises, they are mainly found in flour, fruit and vegetables. However, they also grow on a variety of textiles, for example linen fabrics. Alternaria can trigger allergic reactions such as, for example, running nose, cough, sneezing, urticaria or asthma (mold allergy).

Owing to the disclosure of the amino and nucleic acid sequence, it is possible, with the aid of recombinant techniques, to prepare shorter fragments of the complete sequence recombinantly in bacteria, for example in E. coli, or in higher organisms, for example insect cells, yeasts or eukaryotic cells. It is precisely short polypeptides that can also be provided readily via the chemical route with the aid of solid-phase synthesis.

The present invention furthermore relates to a vaccine which can be employed for desensitizing patients to a mold allergy. In the desensitization, patients who suffer from an allergy are brought into contact with a small amount of an antigen, whereby it is intended that neutralizing IgE antibodies are formed. The antigens with which the patient has come into contact are bound by these neutralizing antibodies. The antigen-antibody binding of antibodies of the IgE type, which trigger allergic reactions, are thereby avoided. The polypeptides according to the invention can therefore be used for preparing a vaccine. To this end, the recombinantly produced, or else chemically produced, polypeptides can be incorporated into a suitable vaccine formulation. In addition to the polypeptides, the vaccine formulation can also comprise conventional additives and formulation auxiliaries, as well as adjuvants.

The present invention also relates to the use of a polypeptide according to the invention for a diagnostic detection of a disease. Such a disease usually takes the form of an allergy. The polypeptides are employed in a suitable diagnostic detection system. This may take the form of a radioimmunoassay (RIA), or preferably also an ELISA (enzyme-linked immunosorbent assay). The usual configuration of such a diagnostic assay is known.

Another aspect of the present invention relates to nucleotides from the nucleotide sequence with the sequence ID No. 2. The nucleotide sequence with the sequence ID No. 2 is likewise shown in FIG. 1. It is part of the gene for the Cladosporium herbarum mannitol dehydrogenase according to the invention.

A further aspect of the present invention relates to polynucleotides with the nucleotide sequence of the sequence ID No. 7. Parts of this nucleotide sequence are likewise the subject-matter of the invention. With the aid of this nucleotide sequence or parts thereof, a desired polypeptide can be produced recombinantly in suitable host cells.

A further aspect of the present invention relates to polynucleotides with the nucleotide sequence, sequence ID No. 12. This is a nucleotide sequence which encodes the Alternaria alternata mannitol dehydrogenase and nucleotide sequences which are immediately adjacent to the coding region. Parts of this nucleotide sequence are also the subject-matter of the present invention.

A polynucleotide according to the invention has at least eight consecutive nucleotides, preferably at least 12, more preferably at least 20 and most preferably at least 50 consecutive nucleotides. For some fields of application, the nucleotides must be longer, in which case the polynucleotides have at least 100 consecutive nucleotides selected from the sequence ID No. 2, ID No. 7 or sequence ID No. 12.

The polynucleotides according to the invention can be used for detecting a mannitol dehydrogenase. It is preferred to detect the presence of a gene encoding this mannitol dehydrogenase from Cladosporium herbarum and/or Alternaria alternata. These methods take the form of nucleic acid amplification methods which are known per se. A suitable example for this purpose is NASBA (nucleic acid sequence based amplification) or, more preferably, polymerase chain reaction (PCR).

Since the nucleic acid sequences encoding the Cladosporium herbarum and Alternaria alternata mannitol dehydrogenase have been disclosed, it is possible to select those nucleotide sequences for the amplification which have a very high degree of homology, or even identity. It can be expected that, when using such highly-specific primers, other mannitol dehydrogenases from related organisms are also amplified since a high degree of homology in the amino acid sequences suggests a high degree of conservation in such a gene region.

Thus, it is preferred to use such highly conserved regions for nucleic acid diagnostics in the case when the antigen is to be isolated not only from Cladosporium and/or Alternaria species, but also from other mold species.

Regions which have a low degree of homology with one another are therefore better suited for fine diagnostics, that is to say for the distinction both between Alternaria and Cladosporium species and for the fine differentiation within Cladosporium or Alternaria species. FIG. 6 shows the coding regions from Cladosporium herbarum and Alternaria alternata together. Identical nucleotide sequences are shown against a black background and identify conserved regions.

Such a method can be used for detecting the presence of the mold Cladosporium herbarum and/or Alternaria alternata. Such applications are of interest not only in medical diagnostics, but also in other fields, for example in the fields of hygiene and food testing. In this context, it must be taken into consideration that Cladosporium herbarum is capable of growth even at relatively low temperatures of up to approximately +6° C. and that it can therefore constitute an undesired contamination in fields of food technology. The detection even of small amounts of Cladosporium herbarum may play an essential role in the control of foods and their quality control.

A further aspect of the present invention is the disclosure of a method for preparing a polypeptide according to the invention. First, a gene from a mold, preferably from Cladosporium herbarum or Alternaria alternata, can be amplified with the aid of the polynucleotides according to the invention and with the aid of the polymerase chain reaction. This polynucleotide can then be incorporated into a suitable vector with which a host cell is transformed. Suitable vectors multiply in the host cell, during which process the polypeptides are expressed. The host cells may take the form of conventional host cells. Suitable for this purpose are bacterial host cells such as Escherichia coli or Bacillus subtilis or yeasts such as, for example, Saccharomyces cerevisiae or Pichia pastoris.

For the purposes of the present invention, IgE immunoblots of Cladosporium herbarum crude extract were assayed with sera from 62 patients. An immunoreactive protein of molecular weight 29 kD, which was recognized by 61% of the patients' sera, was identified. The patients had been preselected in a skin test or blood test (RAST) and showed a positive response to Cladosporium herbarum extract. No other allergen in the Cladosporium herbarum extract reacted with such a high percentage of patients' sera. It is therefore assumed that this protein is the major allergen of Cladosporium herbarum.

The immunoreactive proteins disclosed within the scope of the present invention are important allergens, not only for diagnostic purposes, but also for the therapy of allergens to molds, in particular Cladosporium and Alternaria species. If appropriate, these allergens, together with other allergens, for example Alt a 1 [Unger A. et al. (1999), Clinical testing of recombinant allergens of the mold Alternaria alternate, Int. Arch. Allergy Immunol. 118, 220-221] and Enolase [Simon-Nobbe B. et al. (2000), IgE binding epitopes of enolases, a class of highly conserved fungal allergens, I. Allergy Clin. Immunol. 106, 887-895] can be employed both in diagnostics and for therapeutic purposes.

The two-dimensional separation by isoelectric focusing and SDS-PAGE showed this Cladosporium herbarum protein as a 29 kD spot and at isoelectric point at pH=5.8.

The protein was purified to homogeneity in a conventional method (example 1). The yield amounted to 1 mg. The homogeneously purified protein was then assayed in the IgE immunoblot with a pool of six patients and was highly positive (example 2).

The protein which had been purified to homogeneity was partially sequenced by Edmanic degradation, starting at the N terminus, and internal peptide sequences were determined after degradation with CNBr.

N-terminal and internal peptide sequences were determined after digestion with trypsin by subjecting approximately 50 μg of protein, which had been obtained by excising a spot from the two-dimensional electrophoresis, to Edman degradation.

Table 1 shows a list of all peptide sequences which were identified. The single-letter code was used. The amino acids which are shown against the black background in table 1 were found in the sequence of the Cladosporium fulvum mannitol dehydrogenase. TABLE 1 Peptide sequences of the C. herbarum NADP-dependent mannitol dehydrogenase, N-terminal sequence

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Analytical method: Edman sequencing

a) Starting material: native purified protein b) Analytical method: Edman sequencing Peptide 1

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Analytical method: MS/MS c) Note: overlaps with the N-terminal sequence Peptide 2

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Tryptic digest c) Analytical method: sequencing

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Tryptic digest c) Analytical method: MS/MS Peptide 3

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Tryptic digest c) Analytical method: sequencing Peptide 4

a) Starting material: crude extract separated by means of 2-dimensional SDS gel b) Tryptic digest c) Analytical method: MS/MS Peptide 5

a) Starting material: native purified protein b) CnBr digest c) Analytical method: sequencing

The peptide sequences were compared with all the protein sequences listed in the databases (Swissprot, GenBank and the like) by computer-aided homology search. All peptides showed homology with the family of the mannitol dehydrogenases. The mannitol dehydrogenase with which the peptides show the highest degree of sequence similarity is the Cladosporium fulvum mannitol dehydrogenase. A purification of Cladosporium fulvum mannitol dehydrogenase is described in Noeldner et al., Physiological and Molecular Plant Pathology (1994) pp. 281-289. The position of these peptides in the sequence can be seen in the alignment (Table 2).

Table 2 shows the arrangement of the polypeptides with seq. ID No. 4, 5 and 6 with reference to the homology with C. fulvum.

Owing to these results, the enzyme activity of the protein which had been purified to homogeneity was assayed. The experiments reveal that the highly purified major allergen of Cladosporium herbarum is indeed a mannitol dehydrogenase which catalyzes the following metabolic reaction: Fructose+NADPH+H+

mannitol+NADP⁺. Furthermore, it has been found that NADH is not active as cosubstrate and that fructose-6-phosphate is also not active as substrate. Fructose-6-phosphate has an inhibitory effect on the reaction. The method of the activity determination is described in example 3.

Then, the N-terminal peptide sequence and an internal peptide sequence of the Cladosporium herbarum mannitol dehydrogenase were used for designing PCR primers by means of back translation. The primer selection is compiled in table 3. TABLE 3 DNA sequence of the oligos derived from the peptides Oligo 1: derived from the N-terminal sequence of the C. herbarum mannitol dehydrogenase (MtDH) (see seq. ID No. 2) oligo sequence: 5′ CA (A/G) CA (A/G) GC (I/C) AC (I/C) AA (A/G) CA (C/T) GA 3′ Oligo 2: derived from peptide 4 of the C. herbarum MtDH oligo sequence: 5′ AC (A/G) AA (A/G) TC (A/G) CT (I/C) AG (I/C) CC (A/G) GT (A/G) TC 3′ The primers are mixtures of synthetic oligonucleotides. (A/G) . . . means that both adenine and guanine are found in the oligonucleotides at this position. The same applies to (C/T) and (I/C), where I represents the base inosine.

These primers which are shown in table 3 were used to carry out a PCR reaction with the DNA from a Cladosporium herbarum cDNA library constructed by the inventors (Achatz G et al., 1995, Mol. Immunol., 32; 213-27). The result was a 636 bp band. This band was sequenced by automated DNA sequencing as described by Sanger (1977, Proc. Natl. Acad. Sci. USA, 74; 5463-7) using the PCR primers as sequencing primers. Seq. ID No. 2 was identified in this process. The protein. sequence (seq. ID No. 1) derived from this DNA sequence has 87% identity with the protein sequence of the Cladosporium fulvum mannitol dehydrogenase. If the substitution by chemically related amino acids (for example I-V, isoleucine—valine and the like) is also taken into consideration, this value rises to 92%. With the plausible assumption that the Cladosporium herbarum mannitol dehydrogenase, like the Cladosporium fulvum mannitol dehydrogenase, has a total length of 267 amino acid, as much as 65% of the amino acid sequence of the major allergen (mannitol dehydrogenase) from Cladosporium herbarum were determined by firstly peptide sequencing and secondly DNA sequencing. The total sequence of this protein which is known to date, and the alignment of this sequence with the homologous Cladosporium fulvum sequence, are shown in FIG. 2.

EXAMPLE 1

Protein Purification

1. Ammonium Sulfate Precipitation:

Prefractioning can be achieved by an ammonium sulfate concentration of 50%, with mannitol dehydrogenase (MtDH) remaining in the supernatant.

50 mM Tris-HCl, pH 7.5 were added to the extract. Proteases were inhibited with 1 tablet of Roche Complete per 100 ml of extract and 2 mM EDTA.

The precipitation was carried out by adding solid, ground ammonium sulfate and was carried out in two steps, first 0-30%, then 30-50%. The precipitation was equilibrated for at least 45 minutes before the extract was centrifuged at 12 000 g. The supernatant was filtered and purified further via hydrophobic interaction chromatography (HIC).

2. HIC (Phenyl-Sepharose):

The supernatant from the ammonium sulfate precipitation was brought to pH 6.5 using 3 M sodium acetate. The column (8 ml Source, 15 PHE, PHARMACIA) was equilibrated with 1.2 M ammonium sulfate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA and loaded with the sample at a flow rate of 1 ml/min. After the column had been washed with 20 ml of buffer, it was eluted with a gradient of 1.2 M ammonium sulfate to 0.6 M ammonium sulfate over 40 ml.

The mannitol dehydrogenase (MtDH) fractions were pooled and prepared for the anion exchanger. The volume is concentrated via Centricon centrifuge tubes (Millipore); buffer exchange flow 50 mM Tris-HCl, pH 7.5 with the aid of PD-10 Desalting Columns (AMERSHAM-PHARMACIA).

3. Anionic Exchanger (Q-Sepharose):

The column (8 ml Source 15 Q, PHARMACIA) was equilibrated with 15 mM Tris-HCl, pH 7.5. It was eluted with a 0-300 mM NaCl gradient over 100 ml.

EXAMPLE 2

Immune Blot of the Native Purified MtDH After Separation in the SDS Gel

Native purified MtDH was separated by molecular weight in a reducing SDS gel (Laemmli U K, Nature, 1970; 27:680-5). Subsequently, the protein was transferred onto a PVDF membrane in a Western blot (Towbin H et al., Proc. Natl. Acad. Sci USA, 1979; 76:4350-4). After free binding sites had been saturated (30 minutes in blocking buffer: 50 mM sodium phosphate pH 7.5, 0.5% Tween 20, 0.5% BSA, 0.05% NaN₃), the membrane was incubated with patients' serum (1:10 diluted in blocking buffer). Then, the membrane was washed (with blocking buffer, 3×10 minutes) to remove unspecifically bound antibodies. Specifically bound IgE-Ab were detected with the aid of an ¹²⁵I-labeled rabbit anti-human IgE antibody. After the membrane had been exposed to an X-ray film, the result was available.

Results:

1) The native purified MtDH reacts specifically with the IgE antibodies of C. herbarum allergy sufferers. A prominent IgE-reactive band is revealed at 29 kD.

2) The same result, viz. a prominent IgE-reactive band at 29 kD, is obtained when a C. herbarum total extract is separated in the SDS gel and subsequently incubated with patients' serum in an immune blot.

EXAMPLE 3

Immune Blot of the Native Purified MtDH After 2-Dimensional Separation

Native purified MtDH was separated under denaturing conditions in an isoelectric focusing (O'Farrel P H, J. Biol. Chem., 1975; 250:4007-21) according to the net charge (isoelectric point) of the protein. Thereafter, the protein separated thus was subjected to SDS gel electrophoresis (Laemmli U K, Nature, 1970; 27:680-5), whereby a separation by molecular weight took place in addition. The protein was transferred to a PVDF membrane in a Western blot (Towbin H et al., Proc. Natl. Acad. Sci. USA, 1979; 76:4350-4). After free binding sites had been saturated (30 minutes in blocking buffer: 50 mM sodium phosphate pH 7.5, 0.5% Tween 20, 0.5% BSA, 0.05% NaN₃), the membrane was incubated with patients' serum (1:10 diluted in blocking buffer). Then, the membrane was washed (with blocking buffer, 3×10 minutes) to remove unspecifically bound antibodies. Specifically bound IgE-Ab were detected with the aid of an ¹²⁵I-labeled rabbit anti-human IgE antibody. After the membrane had been exposed to an X-ray film, the results were available:

Results:

1) The native purified MtDH reacts specifically with the IgE antibodies of C. herbarum allergy sufferers. A prominent IgE-reactive spot was observed at a molecular weight of 29 kD and an isoelectric point of 5.8.

2) The same result, viz. a prominent IgE-reactive spot at a molecular weight of 29 kD and an isoelectric point of 5.8 is obtained when a C. herbarum total extract is separated in a two-dimensional gel and subsequently incubated with patients' serum in an immune blot. An IgE-reactive protein with a molecular weight of 29 kD and an isoelectric point of 5.6 is additionally found in the total extract. This protein could be an MtDH isoform.

EXAMPLE 4

To confirm the results according to the invention, the enzyme activity was determined with the traditionally purified protein. The absorption of NADPH was measured in a photometer at 340 nm.

Reaction mix (1 ml):

-   -   50 mM Tris-HCl, pH 7.5     -   0.25 mM NADPH or NADH     -   D-fructose or fructose-6-phosphate (0.1; 0.2; 0.4; 0.6; 0.8;         1.0; 1.2 M)     -   H₂O to 1 ml     -   the reaction is started with 0.5 μl of MtDH

Results:

-   -   Reaction with fructose and NADPH     -   No reaction with fructose-6-phosphate and NADH

EXAMPLE 5

Sequence of Mannitol Dehydrogenase (MtDH)

The complete sequence of the Cladosporium herbarum mannitol dehydrogenase was determined as described hereinbelow.

The peptide sequences obtained by Edman degradation of the Cladosporium mannitol dehydrogenase which had been purified to homogeneity were used to synthesize primer mixtures for the PCR. The PCR resulted in a band of 636 nt which was firstly sequenced and secondly used as hybridization probe for screening our cDNA library. A complete Cladosporium herbarum mannitol dehydrogenase (MtDH) clone was isolated and sequenced. The complete sequence is shown in FIG. 3; it has 84% identity with the published sequence of the C. fulvum MtDH.

Table 4 shows the sequence alignment of the two mannitol dehydrogenases of Cladosporium herbarum and Cladosporium fulvum, only the amino acids which differ being shown.

Amino acid sequences which are shown against the black background mean identical amino acid sequences, chemically similar amino acids are shown against a grey background, and amino acids which differ are shown against the normal background.

The C. fulvum sequence is represented as seq. ID No: 9, and the C. herbarum amino acid sequence as SEQ. ID No: 10.

Table 4 shows the regions of the polypeptide which may be suitable for the detection of or a vaccine for Cladosporium. They are the regions with no differences.

In the regions with pronounced differences it must be presumed that the immunological reactions differ; such regions can therefore comprise highly specific epitopes.

When determining the sequence shown in FIG. 3, it was found that minor differences occurred in the nucleotide sequence in comparison with the originally isolated part-sequences. This can be attributed to differing sequences which were present in the gene library. However, these differences do not affect the present invention in any way. The invention relates to the disclosed differing sequences, since it is assumed that they are variants of the gene.

EXAMPLE 6

Expression in E. coli, and Reactivity with Patients' Serum

The open reading frame of MtDH was cloned into the following expression vectors:

-   -   a) pHis-Parallel 2 Vector (Xhol/BamHI) (Ref.: P. Sheffield, S.         Garrard, and Z. Derewenda (1999). Overcoming expression and         purification problems of RhoGDI using a family of “parallel”         expression vectors. Protein Expr Purif 15,34.)     -   b) pMW172 Vector (Ndel/EcoRI) (Ref.: M. Susani, P. Jertschin, C.         Dolecek, W. R. Sperr, P. Valent, C. Ebner, D. Kraft, R. Valenta,         and O. Scheiner (1995). High level expression of birch pollen         profiling (Bet v 2) in Escherichia coli: purification and         characterization of the recombinant allergen. Biochem Biophys         Res Commun 215, 250.)

The plasmids were subsequently transformed into Escherichia coli strain BL21 (DE3). For the subsequent induction, 5-ml-portions of LBamp were inoculated with 50 μl of a stationary overnight culture of the two clones and the mixtures were shaken at 37° C. until a OD₆₀₀ of 0.8 had been reached (approx. 4 hours). The protein expression was induced with 0.8 mM IPTG. After incubation for 4 hours at 37° C. in a shaker-incubator, the E. coli suspensions were spun down for 15 minutes at 4000 rpm. The bacterial pellets were subsequently resuspended in 1 ml of 1×PBS, and 6-μl-portions of the dissolved bacterial pellets were separated by SDS-PAGE and subsequently stained with Coomassie BBR250. This gave the following results: the E. coli cells which had been transformed with the expression plasmids and induced with IPTG, but not the E. coli cells without plasmid, reveal a pronounced protein band at the molecular weight expected in each case, viz. 30 kD and approximately 33 kD, respectively (apparent molecular weight).

An IgE immune blot was carried out with the polypeptides which had been separated with the aid of a gel. The serum of a Cladosporium-positive allergy sufferer was used. The bound IgE antibodies were detected with the ¹²⁵-I labeled rabbit anti-human IgE antibody (RAST). The two foreign proteins which were overexpressed in E. coli react strongly with the IgE of the patient, but not the proteins of E. coli itself.

EXAMPLE 7

Determination of the Frequency of the Response to Recombinant MtDH with the Aid of 30 Sera of Cladosporium-Positive Allergy Sufferers

The experiment described in example 6 was repeated, but 30 different Cladosporium-positive allergy-sufferer sera which had not been preselected were used. The control revealed a very low immune reactivity of the E. coli extract with the second antibody (RAST). This can probably be attributed to an artifact. As expected, other controls were negative.

Among 30 patients, 20 revealed an IgE-positive band at 30 kD which was more pronounced than the weak band in the control experiment. MtDH is thus recognized by approximately two thirds of the Cladosporium-positive allergy sufferers. This finding is important because this experiment demonstrates that recombinant Cladosporium herbarum MtDH can be employed as diagnostic and therapeutic for the majority of the patients.

EXAMPLE 8

To clone the Alternaria alternata mannitol dehydrogenase, a cDNA bank in Lambda-ZAP (Stratagene, La Jolla, Calif., USA). This cDNA cloned library was prepared with the aid of isolated mRNA from Alternaria alternata.

As described above, the cDNA library was screened with a DNA probe, with initially 24 primary clones being isolated. 5 of these clones were sequenced completely. All 5 sequences were identical in the coding region. The translation of the nucleotide sequence into an amino acid sequence and the comparison with the amino acid sequence of the Cladosporium herbarum mannitol dehydrogenase revealed that the reading frame was complete. The sequence had 74% identity with the sequence from Cladosporium herbarum.

EXAMPLE 9

The open reading frame of the clone encoding the Alternaria alternata mannitol dehydrogenase was then recloned in the expression vector pHIS-parallel 2 [P. Sheffield et al. (1999), Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors, Protein Exp. Purif. 15, 34] using the restriction cleavage sites Bam H I (N-terminally) and Xho I (C-terminally). Upon expression in E. coli BL21 and subsequent analysis of the gene products with the aid of SDS-PAGE gel electrophoresis and Coomassie Blue staining, a pronounced protein band appeared at a molecular weight of approximately 30. kD. This corresponds approximately to the molecular weight which would be expected theoretically.

EXAMPLE 10

The following procedure was chosen for purifying the recombinantly produced protein, which is provided with a poly-His fragment at the C terminus: the E. coli cells with the expression vector, which express the foreign protein, the Alternaria alternata mannitol dehydrogenase, were first lysed in the customary manner. It was found that the recombinantly produced protein was present in insoluble form. The inclusion bodies formed by overexpression of the foreign protein were first solubilized in a buffer with 6-molar urea and subsequently purified by affinity chromatography over a nickel chelate column. The recombinantly produced mannitol dehydrogenase was applied in 6 M urea buffer. Imidazole buffer was employed for the elution. The protein-comprising fractions were subsequently purified further by preparative SDS-PAGE gel electrophoresis and analyzed, during which process it emerged that the allergen was already purified to virtually complete homogeneity. Staining with Coomassie-BB-R only revealed one band with a molecular weight of approximately 30 kD.

EXAMPLE 11

The protein prepared in accordance with example 10 was separated by gel electrophoresis and tested in an IgE immune blot with the sera of 28 patients. All of the 28 sera originated from patients who had shown a positive response to the Alternaria alternata crude extract and who had been pretested both in a skin test and in an RAST. A pronounced band was visible in the immune blot in the case of 9 of the patients' sera tested. This corresponds to approximately 32% of the Alternaria alternata-sensitized patients. 

1-35. (canceled)
 36. A vaccine for desensitizing patients to a mold allergy, comprising at least one polypeptide selected from the group consisting of (a) a polypeptide comprising at least 11 consecutive amino acids from SEQ ID No: 1; (b) a polypeptide comprising at least 11 consecutive amino acids from SEQ ID No: 8; (c) SEQ ID No: 4, (d) SEQ ID No: 5; (e) SEQ No: 6; and (f) a polypeptide comprising at least 11 consecutive amino acids from SEQ ID No:
 11. 37. The method of making a vaccine for desensitizing patients to mold allergy comprising combining the polypeptide of claim 36 with an adjuvant.
 38. The method of making a diagnostic kit comprising a diagnostic agent comprising the polypeptide of claim 36 and a container.
 39. The method of claim 38, wherein said diagnostic agent detects an allergy.
 40. A polypeptide selected from the group consisting of (a) a polypeptide comprising at least 100 consecutive amino acids from SEQ ID No: 1 (b) a polypeptide comprising at least 100 consecutive amino acids from SEQ ID No: 8; (c) SEQ ID No: 4; SEQ ID No: 5; and (d) SEQ ID No:
 6. 41. The polypeptide of claim 40 which has at least one epitope.
 42. The polypeptide of claim 41, wherein said epitope is specific for a mold.
 43. The polypeptide of claim 42, wherein the mold belongs to the genus Cladosporium.
 44. The polypeptide of claim 43, wherein the mold is Cladosporium herbarum.
 45. The polypeptide of claim 40, which comprises SEQ ID NO: 1 or SEQ ID NO: 8 and is mannitol dehydrogenase.
 46. A polypeptide comprising at least 50 consecutive amino acids SEQ ID No:
 11. 47. The polypeptide of claim 46, which has at least one epitope.
 48. The polypeptide of claim 46, wherein the epitope is specific for a mold.
 49. The polypeptide as claimed in claim 48, wherein the mold belongs to the genus Alternaria.
 50. The polypeptide as claimed in claim 49, wherein the mold is Alternaria alternata.
 51. The polypeptide of claim 46-50, which is mannitol dehydrogenase.
 52. A polynucleotide comprising at least 50 consecutive nucleotides of SEQ ID No: 2, SEQ ID NO: 7 or SEQ ID No:
 12. 53. The polynucleotide of claim 52, which comprises at least 100 consecutive nucleotides.
 54. The method detecting a mannitol dehydrogenase comprising using the polynucleotide of 52 in a nucleic acid based amplification system.
 55. The method of claim 54, wherein the mannitol dehydrogenase from a mold.
 56. The method of claim 55, wherein the mold is Cladosporium herbarum and/or Alternaria alternata.
 57. A method of making mannitol dehydrogenase comprising expressing the polynucleotide of claim 52 in a host cell.
 58. A method of detecting mannitol dehydrogenase comprising amplifying DNA using a polynucleotide comprising at least eight consecutive nucleotides from SEQ ID No: 2 or SEQ ID No:7 or SEQ ID No: 12 in a polymerase chain reaction (PCR).
 59. A vector comprising a nucleic acid molecule of claim
 52. 60. A host cell transformed with the vector of claim
 59. 61. The host cell of claim 60, which is Escherichia coli.
 62. The host cell of claim 60, which is Pichia pastoris.
 63. A method of recombinantly producing a protein comprising culturing the host cell of claim 60 under protein expressing conditions and isolating said protein. 